Cells
Cell lines (HOS and U2OS OS cells and hFOB1.19 human osteoblasts) were acquired from the ATCC. The OS lines were grown in DMEM at 37℃ with 5% carbon dioxide, while the hFOB1.19 line was grown in DMEM/Ham’s F12 medium at 34 ℃. All media included 10% fetal bovine serum (FBS) and 1% antibiotics
CCK‑8 assays
Following treatment, OS cells (8×103 per well) were grown in 96-well plates for 0, 24, 48, or 72 h under normal conditions, before adding 10 µL of CCK-8 solution (C6005, New Cell & Molecular Biotech, Shanghai, China) to each well for 2 h. Absorbances at 450 nm were then read in a microplate reader (Bio-Rad, USA).
Transwell assays
Two hundred microliters containing OS cells serum-free DMEM were inoculated in the upper chamber of the Transwell apparatus with/without Matrigel coating (BD Biosciences, San Jose, CA, USA), while 800 µL of DMEM with 10% FBS placed in the lower compartment. After 12 h, the cells remaining in the upper compartment were collected with swabs while those on the bottom surface were fixed with paraformaldehyde (4%), before staining with crystal violet (0.5%), and imaging with a microscope (Zeiss, Germany).
Wound-healing assays
OS cells were grown in 6-well plates until fully confluent. Three parallel scratches were made in the monolayer with a 200 µL micropipette tip, and culture was continued in DMEM/F12 with DMSO or XN. Scratch widths were measured and imaged after 0 and 24 h.
Colony formation assay
Treated OS cells (1×103) were grown in 6-well plates for 10 days, after which they were fixed and stained as above and the colonies were enumerated.
EdU assays
A BeyoClick™ EdU Cell Proliferation Kit (C0075S, Beyotime Biotechnology, Shanghai, China) was used, following the provided directions. Specifically, OS cells were inoculated in 12-well chambers (Ibidi, Germany), and 200 µl of 50µM EdU were introduced to individual wells for 2 h after which cells were fixed as above and treated with Triton X-100 (0.3%, 10 min, RT) for permeabilization. After rinsing, cells were incubated with Hoechst 33342 (5 µg/ml, 30 min) and imaged with a LSM 510 laser-scanning confocal microscope (Zeiss).
RNA-Seq and bioinformatics analyses
Total RNA was extracted from XN-treated (10 µg/ml, 24 h) cells using TRIzol (Thermo, USA). RNA-Seq was undertaken by Shanghai Applied Protein Technology Co., Ltd. (APTBIO, Shanghai, China). Cufflinks calculated the fragments per kilobase per million mapped fragments (FPKM) values of exons and HTSeq-Count was used to determine gene read counts. Differentially expressed genes (DEGs) were identified using the criteria of [log2FC] > 1 and p < 0.05. DEG functions and pathways were explored using GO and KEGG analyses, respectively.
Cell infection
Cells were overexpressed due to lentiviral infection (Sangon Bioengineering, Shanghai, China). When OS cells were in log phase, cells were digested with trypsin. Cells were inoculated in 6-well plates at a density of 105 cells/well. After 24 hours of routine culture, the cell fusion rate reached about 75%, and appropriate amount of packaged lentivirus was added. After 6 hours of infection, fresh medium was added.
q-PCR
The extracted total RNA was reverse-transcribed, as described [6]. Real-time PCR was conducted on a IQ5 Multicolor Real-Time PCR Detection system (Bio-Rad Laboratories, USA), as described [6] using GAPDH as reference. The primer sequences were forward 5’-ACAACTTTGGTATCGTGGAAGG-3’ and reverse 5’-GCCATCACGCCACAGTTTC-3’ for GAPDH; 5’-GCAGGCTACGAGCAAAGTGAAC’ and reverse 5’-GCTTCTGATATCCAGGAGGGCA − 3’ for EFEMP1.
Western blotting
Cell lysis was performed with RIPA buffer and protein levels were assessed using a BCA kit. Forty micrograms of protein were applied to 5–10% SDS-PAGE and electro blotted to nitrocellulose membranes. The blots were blocked (5% skimmed milk, 1 h) and treated (overnight, 4℃) with primary antibodies against GAPDH (ab181602, Abcam), EFEMP1 (ab256467, Abcam), AKT (4685, CST), p-AKT (4228, CST), PI3K (4249, CST), and p-PI3K (4228, CST). After incubation with secondary antibodies (ZB-5301, ZSGB-BIO, China) (1 h, RT), enhanced chemiluminescence was used to visualize the bands.
Immunofluorescence assays
Cells on coverslips in 6-well plates were treated before fixation for 30 min as above and incubation with 0.1% Triton X-100 (10 min) before treatment with primary antibodies (overnight, 4 ℃), secondary antibodies (60 min, RT), staining with DAPI, and imaging under confocal microscopy (FV10i, Olympus, Japan).
In vivo experiment
Approximately 100µL of 1×107 HOS cells in DMEM were injected subcutaneously into the dorsal surfaces on mice. Tumor sizes were monitored every five days using vernier calipers and the volume was determined as (V) = length*width2/2. The effectiveness of treatments was assessed by the growth and final weights of the tumors.
Immunohistochemistry (IHC)
Tumor samples were embedded in paraffin, followed by dewaxing with xylene and rehydration in an ethanol gradient. After antigen retrieval (sodium citrate, pH 6.0, 15 min), the sections were sealed using serum and probed with primary antibodies (overnight, 4°C) against Ki-67 (ab15580, Abcam) and PCNA (2586, CST). The sections were rinsed with PBS and treated with secondary antibodies (10 min, RT) followed by DAB Plus developer (1–2 drops in 1 mL DAB Plus substrate, 1–5 min and imaged (Nikon, Ni-u). Cells positive for Ki-67 and PCNA were enumerated in three randomly selected fields.
Hematoxylin and eosin (H&E) staining
Paraffin tissue sections (5 µm) were stained with H&E (Abcam) and imaged under light microscopy (Zeiss).
Statistical analysis
Data were analyzed using SPSS 17.0 and graphs were compiled with GraphPad Prism 9. Between-group comparisons were examined by one-way ANOVA. Data are presented as mean ± standard deviation (SD), with P < 0.05 representing significance.